PROJECT SUMMARY The goal of our Program renewal is to build upon the novel and unexpected discoveries of our last grant period to dissect, perturb, and control molecules and networks that enable or restrict the conversion of a somatic cell to pluripotency. Our last grant period revealed that, during conversion to pluripotency, resident enhancers in somatic cells that maintain cell differentiation must be decommissioned and pluripotency enhancers must be gradually activated, heterochromatic domains must be disassembled, and the post- transcriptional processes of polyadenylation and protein sumoylation must be altered. By investigating these parameters collectively in the next grant period, the Plath, Zaret, and Hochedlinger labs will determine how gene-regulatory networks integrate with cell biological transitions that are crucial for timely and efficient cellular reprogramming. Our three specific projects address the following fundamental questions: 1) How are enhancer patterns reorganized during the conversion of somatic cells to pluripotency, which genomic and epigenomic features are important, and how can heterochromatin be disassembled? 2) What comprises heterochromatin and how can its constituents be antagonized to facilitate reprogramming? 3) How do post-transcriptional regulation of mRNA polyadenylation and protein sumoylation suppress the pluripotency program? Answers to these mechanistic questions will provide insights to facilitate specific avenues to enhance cellular reprogramming to pluripotency as well as direct reprogramming from one somatic fate to another. The principles we uncover will also have direct applications for understanding cell type conversions in development, disease, and regeneration. Notably, new discoveries and scientific overlap between the three P.I.'s makes this a far more synergistic and interdependent proposal than the previous submission. The Program also takes advantage of extensive collaborations between our three groups, all leaders in the field of reprogramming, with a track record of working and publishing together. Our plan to evaluate chromatin and transcriptional features and post-transcriptional mechanisms within one Program will greatly enhance a sophisticated and cohesive view of how somatic cells can change fate to become pluripotent, identify commonalities and potential differences between human and mouse models, and allow us to determine how diverse features can be modulated coordinately to boost the efficiency and faithfulness of reprogramming to pluripotency and of one somatic fate to another. An Administrative Core will ensure efficiency and data sharing, leverages existing cores at our respective stem cell institutions, offer a pilot program to bring in additional investigators supporting our goals, and integrates a Scientific Advisory Board, and make sure that our goals are achieved collaboratively.